This project has two primary aims. The first aim is to understand how proteins which are secreted or inserted into membranes are targeted to transport sites in the endoplasmic reticulum (ER) or, equivalently, the bacterial inner membrane (IM). In particular we are investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its ER-bound receptor in this process. Previous studies have shown that SRP recognizes nascent polypeptide chains containing "signal sequences" and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor. We plan to elucidate the mechanism by which SRP recognizes the highly diverse family of signal sequences found on different proteins and then releases them at the surface of the ER in a regulated fashion. By studying this problem we expect to obtain insight into the function and regulation of proteins that possess broad substrate specificity. The second aim of the project is to elucidate the function of a universal protein conducting channel or "translocon" that is found in both the ER and the IM. We are particularly interested in understanding how the translocon catalyzes two related but seemingly distinct processes, namely the complete transfer of presecretory proteins across a membrane and the integration of membrane proteins into a lipid bilayer. In addition, we would like to determine why the core of the translocon is evolutionarily conserved, but the peripheral subunits are not. We expect that these experiments will provide significant insight into the function of membrane-bound channels. In recent work we have focused on the function of E. coli homologs of SRP, the SRP receptor and the translocon using a combination of biochemical and genetic methods. The function of bacterial SRP has been of particular interest in light of substantial evidence that bacteria utilize predominantly chaperone-based mechanisms to target secreted proteins to the IM. A few years ago we discovered that E. coli SRP plays a crucial role in targeting inner membrane proteins (IMPs) to the IM. Although our work demonstrates that bacteria contain multiple protein targeting pathways, the mechanism by which secreted proteins and membrane proteins are routed into separate pathways is poorly understood. Recent experiments have shed light on this intriguing question. We have found that under physiological conditions the targeting pathway of a protein is dictated by the composition of its targeting signal. Replacement of the signal peptides of two secreted proteins with the first transmembrane segment of an IMP rerouted both proteins into the SRP targeting pathway. A more modest alteration of the signal peptide that simply increases its hydrophobicity also promoted SRP binding. These results imply that different classes of E. coli proteins are targeted by distinct pathways because bacterial SRP binds to a more restricted range of targeting signals than its eukaryotic counterpart. Furthermore, we have also recently obtained evidence that help explain why the SRP pathway has been universally conserved throughout evolution. Surprisingly, we found that SRP depletion only partly blocks the integration of membrane proteins under some growth conditions. The mislocalized IMPs, however, appear to produce toxic aggregates. Our data suggest that by ensuring efficient IMP insertion, SRP protects cells from the deleterious effects of IMP mislocalization. Finally, in a separate study, we have obtained evidence that different molecular chaperones vary in their ability to preserve the translocation-competence of secreted proteins in the cytoplasm.